The Study On Protein Interacting With Actinobacillus Pleuropneumoniae Apx â…  And A Multiplex RT-PCR To Diagnose Swine Reproductive Disorder

The thesis describes two experiments.1 Screening and identification host cellular protein interacting with Actinobacilluspleuropneumoniae Apxâ… In Chinese pig industry, Porcine Contagious Pleuropneumonia (PCP), caused byActinobacillus pleuropneumoniae, is one of the most important porcine respiratorydiseases. The disease is, characterized by fibrous pneumonia and pleurisy and the clinicalsigns vary from high mortality to chronic, retarded growth, and the feed reward rate islow, leading to the severe economic losses in the swine industry worldwide. Apxâ… themost important RTX toxins that are producted by APE However, the pathogenesismechanism of Apxâ… at protein level has not been understood clearly yet. Therefore, thediscovery of the protein that can interact with Apxâ… will be benefit for preventing andcontrolling the disease, simultaneously providing an important model for research ontoxins of other Gram-negative bacterium.Due to the secretion of only Apxâ… by App serotype 10, in this experiment, theserotype was selected to inoculate to TSB medium containing NAD at a finalconcentration of 0.01% and 10mM CaCl₂ followed by incubation for 4-5 hours. Thesupernatants was subjected to ammonium sulfate precipitation for extraction of nativeApxâ… and dialysis against ammonium sulfate. The toxin was concentrated by Sucrose andpurified by using sephadex 200. The protein concentration of toxin was determined byUV spectrophotometer analysis. The hybriddoma that secreting monoclonal antibodiesagainst Apxâ… was inoculated intraperitoneally into Balb/C mice. The ascites washarvested after seven days and IgG was purified through MAbTrap Kit, which wasconfirmed by SDS-PAGE analysis. The good reactivity of native Apxâ… was showed bywestern bolt using mAb against Apxâ… as primary antibody and HRP-conjugated goatanti-mouse IgG as secondary antibodies. The microtiter plates were coated with thenative Apxâ… that was diluted to the concentration of 100μg/ml with 0.1M NaHCO₃ (pH8.6). Then, the phage library was incubated with native Apxâ… in 96-well plates. Theunbinding phages were washed away and the eluted phages were further propagated forthe next round of screening in order to enrich the interesting phages. After four rounds ofscreening, the eluted phages were used to infect E.coli 2738. Eighteen phage clones werechosen from around 100 clones whose bonding ability to target proteins were analyzedthrough ELISA by using native, recombinant Apxâ… A, Apxâ… CA as antigen. Among them, the genomes of 10 clones were extracted and were selected to sequence analyses. Thecomplete agreement and high homology with Sus scrofa cytochrome oxidasesubunit I sequence among the six phage clones was demonstrated while the remainingfour phage clones shared the homology with the following peptides, respectively. Thesepeptides were cadherin EGF LAG seven-pass G-type receptor 3, Rho-GTPase-activatingprotein 6 and Canis familiaris Glutathione S-transferase theta 2, respectively. However,the sequence of one phage clone can not be matched with any sequences in NCBIdatabase. Finally, the binding of the Apxâ… with the ovine cytochrome oxidasesubunit I was confirmed by co-immunoprecipitation. The results paved the way for thefurther research on the molecular mechanism of Apxâ… .2 Development of a Multiplex RT-PCR to detect classical swine fever virus (CSFV),porcine reproductive and respiratory syndrome virus (PRRSV) and Japaneseencephalitis virus(JEV) and preliminary applicationInfection of swines with classical swine fever virus (CSFV), porcine reproductiveand respiratory syndrome virus (PRRSV) and Japanese encephalitis virus(JEV) results inreproductive problem, and multiple infection has been presented for recent years in thepig farms. Precise detection of pathogens and utilization of the effective measures will bebenefit for controling those kinds of diseases. At present, there are some diagnosticmethods, such as specific-gene RT-PCR, monoclonal antibody-based antigen detectionmethod (Sandwich ELISA and Gold immunochromatography) for single pathogen. Thesemethods are playing a vital role in diagnoses of above diseases, but they can notsimultaneously examine concontaminant pathogens. Because CSFV, PRRSV and JEVbelong to the family of RNA viruses, RNA was extracted from the samples containingtargete viruses and used for development of a multiple RT-PCR with designed specificprimers. CSFV, PRRSV and JEV alone or co-infection could be detected in one reaction.The new method will be a powerful tool for disease diagnosis.In this experiment, three sets of primers were designed, respectively, targeting to theconserved sequences in E2 gene of classical swine fever virus (CSFV), a domainencompassing partial ORF6,ORF7 and 3-terminus UTR of porcine reproductive andrespiratory syndrome virus (PRRSV) and E gene of Japanese encephalitis virus(JEV).The expected sizes of RT-PCR amplicons were 288bp for CSFV, 430 bp for PRRSV and1015 bp for JEV, respectively. RNA was extracted from pure culture of the viruses,followed by the synthesis of cDNA that was used as templates for method development.Through optimization by using three sets primers simultaneously, the detection limit ofRNA template in the RT-PCR was 0.27ng for CSFV, 0.049ng for PRRSV and 0.067 for JEV. No false positive results were produced in the case of transmissible gastroenteritisvirus (TGEV), porcine parvovirus (PPV), pseudorabies virus (PrV) and porcinecircovirus type 2 (PCV-2). There were 174 samples, including sera, fetuses and lungtissues which were collected from the pigs that mainly presented high fever, weresubjected to our in-house RT-PCR. PRRSV was detected in these samples with higherdetection rate than CSFV and JEV. Among 174 clinical samples, 53 werePRRSV-positive(30.4%), 8 were CSFV -positive(4.6%), 3 were JEV-positive(1.7%), 3were co-infection of CSFV and PRRSV (1.7%). In order to confirm the reliability of themultiplex PCR, We selected PCR products of CSFV-positive JEV-postive andPRRSV-positive respectively that detected clinical samples by multiplex PCR. Aftersequences analysis and BLAST, it is showed that homologies between PCR products ofCSFV,PRRSV,JEV and target genes sequences were 92%, 90% and 89%, respectively.Those results proved that multiplex RT-PCR have applied value in practices.